Ras molecules are highly conserved signaling elements that mediate cellular responses to a variety of extracellular signals. Ras pathways have been extensively studied in mammalian systems and implicated in the pathogenesis of many human malignancies. Among microorganisms, Ras elements regulate differentiation events including fungal yeast-mycelium transitions. The basidiomycete Cryptococcus neoformans provides a powerful genetic model system in which to dissect the molecular mechanisms of Ras signaling in a human pathogen. Dr. Alspaugh has previously demonstrated that Ras pathways in this organism control hyphal differentiation through a MAP kinase pathway, agar adherence through a cAMP pathway, and high temperature growth by a temperature-dependent regulation of actin polarization. In this proposal, the Alspaugh laboratory will use several unbiased genetic methods to further characterize the Ras pathways in C. neoformans that regulate high temperature growth and cellular differentiation. First, proteins that physically interact with Ras1 will be identified by a yeast two-hybrid screen. It is expected that genes encoding both upstream and downstream components of the Ras1 signal transduction pathway will be identified by this method. Additionally, downstream components of Ras proteins that regulate growth at elevated temperature will be identified by two complementary approaches. Positive effectors of Ras1 signaling will be identified by multicopy suppressor analysis. Inhibitory or regulatory proteins involved in Ras1 control of high temperature growth will be identified using random insertional mutagenesis in the ras1 mutant background and screening for the restoration of high temperature growth. Finally, in collaboration with other mycology laboratories at Duke University, the Alspaugh lab is constructing gene microarrays based on the C. neoformans genome project. Genes that are transcriptionally regulated by Ras1 will be identified using these gene microarrays. The biological relevance of genes identified by any of these approaches will be assessed by gene disruption and analysis of the resulting mutant strains both in vitro as well as in vivo in animal models of cryptococcosis.